What is a latex bead?

A spherical polymer particle in the colloidal size range, latex beads are formed from amorphous polymer (usually polystyrene).

The average molecular weight of the polymer chains in the particle is about 1x106g for particles with diameter <100 nm and drops to about 2.4x105g for larger particles. A polystyrene chain is a linear hydrocarbon chain with a benzene ring attached to every second carbon atom.

The aromatic rings control the way the chains coil and entangle and dominate the space; when a model of the particle surface is viewed all that can be seen is randomly stacked benzene rings with an occasional chain end
sticking out.

Thus the surface is very hydrophobic in character and provides for strong physical adsorption of molecular species with hydrophobic regions. Surfactants and protein molecules stick strongly by simple passive adsorption.

This figure shows a section of the surface of a polystyrene microsphere. The view looks down onto the surface above a sulfate group. Note how the benzene rings dominate the field and present a markedly hydrophobic surface ideal for the adsorption of materials such as proteins.

The ends of the polymer chains usually consist of charged groups and provide the colloid stability of IDC’s UltraClean latex microspheres and prevent them from aggregating. The charged ends take up between 5% to 10% of the particle surface area, allowing about 90% free for placing other molecular species such as the antibodies used in latex agglutination tests.

Why UltraClean microspheres?

IDC’s UltraClean latex microspheres do not need surfactants to prevent aggregation. The clean surfaces take the guesswork out of adsorption and stability experiments.

Surfactant purity is not of a very high standard. There is usually a large variation between supplies from different manufacturers as well as there being a significant batch to batch variability. This inevitably leads to a variation in behavior when it comes to protein attachment.

An additional problem—manufacturers which use surfactants are usually reluctant to inform the end user which surfactants were used and how much remain in the latex dispersion. It is a frequent occurrence with nonionic surfactants that some of the surfactant can be covalently grafted to the particle surface. In this case neither extensive dialysis nor ion exchange resins can remove the material.

It is advisable to eliminate these problems by working with surfactant-free systems. A clean, well-characterized surface will enable you to optimize your assay.

Colloidal Stability Considerations

Latex microspheres are supplied in a well-dispersed form in aqueous suspensions. They aggregate in the absence of any specific cause, such as antibody-antigen interactions, because of van der Waals’ forces acting between the particles. The effect is additive—with a colloidal particle made up of a large number of atoms, the distance over which particles “feel” an attraction to each other can be up to 0.5 µm.

Attraction between two latex beadsThis graph shows how the work required to separate two particles becomes large when polymer microspheres get close together. Smaller particles attract each other less and larger particles more. The attraction is in proportion to the particle size. The thermal energy that is available to help them separate is just 1kT which is the energy from the surroundings. They clearly need a barrier which will keep them apart. UltraClean particles from IDC make use of the electrical charge that we build onto the surface of the particles during their synthesis. Other manufacturers rely on an unspecified blend of surfactants.

Repulsion between two latex beads The electrostatic repulsion in our particles is sufficiently strong that the particles “feel” the repulsion over distances similar to those of the attraction. Repulsion is calculated from the electrical potential due to the charges close to the surface; this potential is known as the ζ-potential. It can be measured experimentally or estimated from the titrated charge with hydrophobic particles.

Total interaction between two latex beads
The total interaction is calculated by simply adding the attraction to the repulsion. At 1 mM salt there is a very large barrier to particles coming into contact. When the salt concentration is increased to 100 mM, the barrier is much lower, but still just enough for stability. At salt concentrations as high as 500 mM, the particles will aggregate. At this high salinity, ionic surfactants are unable to maintain stability and non-ionic ones are required. In the case of hydrophilic latex such as the CML category, there is an additional barrier to aggregation due to the “fuzzy” surface layer made up from the soluble polymer species anchored to the surface. This layer is really a concentrated polymer solution. When two layers are pushed together, the local osmotic pressure increase is high enough to resist significant compression.

Total interaction with electrosteric repulsion
In addition to this strong but short-range effect, electrostatic repulsion starts at the outermost edge of this “fuzzy” layer. The net result of this shift in origin of the electrostatics is an enhanced repulsion. This type of stabilization is electrosteric stabilization and is very robust.

This type of interaction is often found in nature and the biological systems making use of it use proteinaceous surface layers. Fat droplets in milk are erythrocytes are examples.

The dimensions of the “fuzzy” layer are a function of both pH and salt concentration. As the pH is increased, the carboxyl groups within the layer become more and more dissociated and so the layer expands. As the salt concentration is increased, the charges are progressively screened from each other and the layer shrinks back. But at very high salt concentrations the solubility of the polymer layer decreases and it will collapse completely. Aggregation of the particles can now occur and we have reached the critical coagulation concentration. Typically for a hydrophilic latex this would be in excess of 1M sodium chloride making these particles easier to work with in physiological strength buffers.

Sedimentation response

A single polymer microsphere sediments according to Stokes’ law. This relates the sedimentation rate to the density difference between the particle and the liquid, the particle size, the gravitational force and the viscosity of the liquid.

The problem is that any of these can be varied as well as the concentration of particles. Quite quickly after working with polymer microspheres, it becomes clear that we can easily centrifuge 1 ìm particles but we have a much more difficult time with 0.1 ìm. So we must keep in mind each of the above factors in addition to recognizing that the particles are also always in motion.

Lets look at each of these in turn:

The density of polystyrene is 1.055 g/ml, close to water at normal laboratory temperatures. Temperature changes will not alter this very much as polystyrene has a very small coefficient of expansion. Water does change significantly—if we centrifuge at say 4°C the particle will sediment at 95% of the rate at laboratory temperature. In addition, density is also a function of salt concentration. So at a physiological concentration of PBS, the sedimentation rate can slow by up to 30%.

Viscosity is even more sensitive to temperature than the density and at 4°C the sedimentation rate will be slowed further to 54% of the rate at say 25°C.

Sedimentation rates slow as the particles get smaller. For example, when we decrease the particle size by ten times from 1 µm to 0.1 µm, the sedimentation slows to 1% of the value of the bigger particle. This means that instead of centrifuging for 5 minutes for example, we would have to centrifuge for about 8.5 hours!

The other problem with size is that the particles are not static; they diffuse in a random fashion due to the local density fluctuations. This Brownian motion is easily observed with an optical microscope. Why should we worry about this? This random motion opposes the sedimentation.

When we increase the g-force in our centrifuge by increasing the speed the force increases as the speed squared. The curve below shows typical rates for a simple sulfate polystyrene microsphere.

Typical g-force for latex bead sedimentation to be ~1cm/min
CML microspheres have a larger hydrodynamic size than sulfate particles and this size is also a function of salt concentration and pH. Because of this it is less easy to predict the sedimentation rate without closely specifying the system.

The attachment of proteins also alters the rate and this can either increase or decrease the rate depending on the initial particle system. However as the colloid stability is altered, coated particles are usually easier to redisperse quickly and fully.

Selection of particles

The selection of the type of microsphere is dictated by both the type of test and the details of the protein attachment you wish to use. For example if covalent coupling is to be used, then a SuperActive particle should be chosen and the particle size is governed by the test mechanism.

Size issues

Simple visible test: The factors to consider are good visibility, rapid response which is affected by the diffusion rate, and available surface area. A size of 0.3 µm to 0.5 µm is good for visibility and the diffusion is rapid. Also, the area for protein adsorption is relatively large. However, washing by centrifugation is difficult unless high density particles are used. The ease of washing, especially with covalently coupled systems where excess materials should be removed, means that particle sizes in excess of 1µm are frequently used.

Strip or membrane tests: These require particles with a high diffusive mobility through the network. Hence sizes around 0.25 µm are good candidates.

Optical detection tests: Turbidimetric tests usually require particles close to 0.15 µm in diameter at the upper limit. At sizes below this, aggregation marketed increases light scattering. The scattering of light is proportional to the volume squared; a doublet will scatter four times as much as a singlet. The result is that small particles can be used at reasonable solids concentrations, that is, between 0.1% and 1%.

Other automated detection systems are designed around light scattering from much larger particles. These use particles from 1.5 µm to 5 µm and can be designed around the scattering from single particles. With such large sizes, the number of particles per ml is quite small at 1% solids. A 5 µm particle at 1% solids has 6x108 particles per ml (compared to a 500 nm particle with 6x1011 and a 50 nm particle with 6x1014).

Attachment issues

The method of protein attachment is the other important consideration. The orientation and conformation of antibodies can dictate which method is most suitable.

If physical (passive) adsorption is satisfactory, then the most common particle used is a sulfate microsphere. With this type of surface, about 5-10% is occupied by well-separated, monovalent sulfate groups. The remaining 90-95% of the surface is made up of stacked benzene rings of the polystyrene. This is a very hydrophilic surface and allows ample adsorption sites for the hydrophobic sections of protein molecules.

Covalent coupling via a two-step process is usually carried out using a carbodiimide to give an active ester intermediate. The attachment to the particles is through carboxylic acid groups and primary amine groups. If the amine on the protein is to be linked to the surface, then a carboxyl modified latex is selected. Alternatively, the carboxyl groups on the protein can be used as the loci of the linking and then a particle with an aliphatic amine surface is selected.

Covalent coupling by a simple one-step incubation can readily be carried out. Here the primary amino groups on the protein are utilized and there is a choice of two types of SuperActive particles available. The aldehyde/sulfate and the chloromethyl particles both react readily to give effective covalent links. The aldehyde/sulfate particles are more hydrophilic than the chloromethyl particles and this may aid stability. the chloromethyl surface may be easier to block excess sites with small molecules such as glycine.